Q.List the enzymes/proteins acting at a DNA replication fork (helicase, topoisomerase, SSBs, primase, DNA polymerase, DNA ligase) and state the specific role of each.

Topoisomerase relieves the torsional/supercoiling stress generated ahead of the replication fork by DNA unwinding.

Working

During DNA replication, the double helix must be continuously unwound at the replication fork by the enzyme helicase so that each strand can serve as a template. Because DNA is a helical molecule, unwinding one region causes the DNA ahead of the fork to become progressively over-wound and twisted on itself (positive supercoiling), building up torsional stress that would otherwise stall the replication machinery if left unrelieved. Topoisomerase solves this problem: it makes a transient, controlled cut in one strand (Type I topoisomerase) or both strands (Type II, e.g. DNA gyrase in bacteria) of the DNA, allows the DNA to rotate/swivel around the cut to release the accumulated torsional strain, and then reseals (re-ligates) the strand(s), restoring an intact but now relaxed DNA molecule. The o …

During DNA replication in E. coli, DNA-dependent DNA polymerase adds nucleotides only in the 5′ → 3′ direction (it needs a free 3′-OH to extend). This is why replication is continuous on the leading strand and discontinuous (Okazaki fragments) on the lagging strand.

DNA polymerase can add a new nucleotide only to the free 3′-OH end of the growing chain, so synthesis proceeds strictly 5′ → 3′. Because the two template strands are antiparallel and the polymerase moves only 5′ → 3′:

• the leading strand is synthesised continuously toward the fork, while
• the lagging strand is made discontinuously as short Okazaki fragments later joined by DNA ligase. …

Single-Strand Binding proteins (SSB) bind and coat the unwound single DNA strands at the replication fork, keeping them separated (preventing reannealing) and protecting them until they are copied.

During DNA replication:

• Helicase unwinds/separates the double helix at the fork (it does not protect the strands).
• SSB (Single-Strand Binding) proteins then bind the exposed single strands, stabilising the fork and preventing the two strands from re-pairing (reannealing) and from being degraded, keeping them available as templates. …

Deoxyribonucleoside triphosphates (dNTPs) serve two roles in replication: (I) they are the substrates (building blocks) added to the growing DNA chain, and (III) hydrolysis of their two terminal phosphates supplies the energy for the polymerisation reaction.

During DNA replication, dNTPs (dATP, dGTP, dCTP, dTTP) perform a dual function:

• (I) Act as substrate: the deoxyribonucleotide is incorporated into the new strand as a building block. …

The 3' to 5' exonuclease activity gives DNA polymerase its proofreading/editing function, ensuring highly accurate DNA replication.

DNA polymerase synthesises the new strand in the 5' to 3' direction, but it also possesses a 3' to 5' exonuclease activity that acts in the reverse direction as a proofreading mechanism.

• If an incorrect (mismatched) nucleotide is added to the growing strand, the polymerase detects the resulting distortion, reverses briefly, and the 3' to 5' exonuclease activity excises the wrong nucleotide.
• Only after the error is removed does synthesis resume in the 5' to 3' direction with the correct nucleotide. …

(a) Single strand binding protein.

Single strand binding proteins (SSBPs) coat the separated DNA strands during replication, stopping them from re-annealing or recoiling.

During DNA replication, the enzyme helicase unwinds and separates the two parental strands at the replication fork. Because separated single strands are unstable and tend to recoil back into a double helix or form secondary hairpin structures, single strand binding proteins (SSBPs) bind along the exposed single-stranded DNA, keeping it stretched out and stable so that DNA polymerase can accurately use it as a template. Reverse transcriptase synthesise …

(d) Complementary strand — because the two strands of DNA follow strict base-pairing rules, the sequence of one strand automatically determines (is complementary to) the sequence of the other.

The two strands of DNA are complementary strands, following Watson-Crick base pairing.

DNA is a double helix of two antiparallel polynucleotide strands held together by hydrogen bonds between specifically paired bases: adenine always pairs with thymine (2 H-bonds), and guanine always pairs with cytosine (3 H-bonds). Because of this strict, predictable pairing rule, if the base sequence along one strand is known, the base sequence of the other strand can be deduced exactly — each strand is said to be 'complementary' to the other. This complementarity is also what allows e …

A full round of E. coli chromosome replication takes about 40 minutes.

The E. coli chromosome (~4.6 x 10^6 base pairs) replicates bidirectionally from a single origin of replication (oriC), with the two replication forks moving towards each other and meeting at the terminus roughly opposite the origin. Under standard laboratory growth conditions this single round of replication takes about 40 minutes to complete, even though E …